Vascular calcification is the major cause of calciphic uremic arteriolopathy, idiopathic infantile vascular calcification, and bioprosthetic valve failure, as well as a major finding in pathologies including atherosclerosis, aortic stenosis, calciphylaxis, and medial arterial calcification otherwise known as Monckeberg's sclerosis. Growing evidence links vascular calcification to increased risk of cardiovascular morbidity and mortality in high -risk patients including those with chronic kidney disease (CKD). Thus, efforts to understand the regulation of vascular calcification may lead to improved prevention as well as treatment of this problem. The overall hypothesis of this proposal is that anti-calcific regulatory mechanisms mediated by vascular stromal and inflammatory cells normally predominate in the vasculature, thereby preventing ectopic mineralization. However, under certain pathological conditions, including hyperphosphatemia, pro-calcific mechanisms can overwhelm anti-calcific mechanisms, thereby leading to ectopic calcification. In the previous grant period, we identified novel mineral-inducing and mineral-inhibiting mechanisms of vascular calcification. In Specific Aim 1, we will determine the role of the sodium dependent phosphate cotransporter, Pit-1, in phosphate loading of matrix vesicles, phenotype transition, gene expression, and mineralization of smooth muscle cells (SMCs) in vitro and in vivo. In Specific Aim 2, we will determine the mechanism by which of Cbfa-1 inhibits SMC mineralization in vitro, and the role of Cbfa-1 in vascular calcification in vivo. Specific Aim 3 will determine the mechanism by which osteopontin promotes regression of ectopic calcification in vivo and in vitro.